Chromosomal DNA damage can be a result of several processes and agents of endogenous or exogenous origin. These cause strand breaks or oxidized bases that lead to strand breaks, which relax the normally supercoiled genomic DNA and increase its electrophoretic mobility. The extent of DNA damage can be assessed by single cell gel electrophoresis, where the chromosomal DNA migration distance correlates with the extent of DNA damage. This technique has been used for a variety of applications with several organisms, but only a few studies have been reported for Saccharomyces cerevisiae. A possible reason for this absence is that low cellular DNA content could hamper visualization. Here we report an optimization of the comet assay protocol for yeast cells that is robust and sensitive enough to reproducibly detect background DNA damage and oxidative damage caused by hydrogen peroxide. DNA repair was observed and quantified as diminishing comet tail length with time after oxidative stress removal in a process well described by first-order kinetics with a tail length half-life of 11 min at 37• C. This is, to our knowledge, the first quantitative measurement of DNA repair kinetics in S. cerevisiae by this method. We also show that diet antioxidants protect from DNA damage, as shown by a three-fold decrease in comet tail length. The possibility of assessment of DNA damage and repair in individual cells applied to the model organism S. cerevisiae creates new perspectives for studying genotoxicity and DNA repair.

Pain sensation and aversive behaviors entail the activation of nociceptor neurons, whose function is largely conserved across animals. The functional heterogeneity of nociceptors and ethical concerns are challenges for their study in mammalian models. Here, we investigate the function of a single type of genetically identified C. elegans thermonociceptor named FLP. Using calcium imaging in vivo, we demonstrate that FLP encodes thermal information in a tonic and graded manner over a wide thermal range spanning from noxious cold to noxious heat (8 C-36 C). This tonic-signaling mode allows FLP to trigger sustained behavioral changes necessary for escape behavior. Furthermore, we identify specific transient receptor potential, voltage-gated calcium, and sodium ''leak'' channels controlling sensory gain, thermal sensitivity, and signal kinetics, respectively, and show that the ryanodine receptor is required for long-lasting activation. Our work elucidates the task distribution among specific ion channels to achieve remarkable sensory properties in a tonic thermonociceptor in vivo.

Understanding how the nervous system bridges sensation and behavior requires the elucidation of complex neural and molecular networks. Forward genetic approaches, such as screens conducted in C. elegans, have successfully identified genes required to process natural sensory stimuli. However, functional redundancy within the underlying neural circuits, which are often organized with multiple parallel neural pathways, limits our ability to identify ‘neural pathway-specific genes’, i.e. genes that are essential for the function of some, but not all of these redundant neural pathways. To overcome this limitation, we developed a ‘forward optogenetics’ screening strategy in which natural stimuli are initially replaced by the selective optogenetic activation of a specific neural pathway. We used this strategy to address the function of the polymodal FLP nociceptors mediating avoidance of noxious thermal and mechanical stimuli. According to our expectations, we identified both mutations in ‘general’ avoidance genes that broadly impact avoidance responses to a variety of natural noxious stimuli (unc-4, unc-83, and eat-4) and mutations that produce a narrower impact, more restricted to the FLP pathway (syd-2, unc-14 and unc-68). Through a detailed follow-up analysis, we further showed that the Ryanodine receptor UNC-68 acts cell-autonomously in FLP to adjust heat-evoked calcium signals and aversive behaviors. As a whole, our work (i) reveals the importance of properly regulated ER calcium release for FLP function, (ii) provides new entry points for new nociception research and (iii) demonstrates the utility of our forward optogenetic strategy, which can easily be transposed to analyze other neural pathways.

Many extracts prepared from plants traditionally used for medicinal applications contain a variety of phytochemicals with antioxidant and antigenotoxic activity. In this work we measured the DNA protective effect of extracts of Ginkgo biloba leaves from oxidative stress using Saccharomyces cerevisiae as experimental model. The extract improved viability of yeast cells under oxidative stress imposed by hydrogen peroxide. In accordance with previous reports on antioxidant properties of G. biloba extracts, pre-incubation of yeast cells promoted a decrease in intracellular oxidation. We assessed DNA damage by our recently developed yeast comet assay protocol. Upon oxidative shock, DNA damage decreased in a dose-dependent manner in experiments of pre-incubation and simultaneous incubation with the extract, indicating a direct protective effect. In addition, the extract improved DNA repair rate following oxidative shock as measured by faster disappearance of comet tails. This suggests that the extract stimulates the DNA repair machinery in its DNA protective action in addition to directly protect DNA from oxidation. The observed DNA repair depends on the DNA repair machinery since no DNA repair was observed under restrictive conditions in a conditional mutant of the CDC9 gene (Accession No. Z74212), encoding the DNA ligase involved in the final step of both nucleotide and base excision repair.

Ryanodine receptors (RyR) are essential regulators of cellular calcium homeostasis and signaling. Vertebrate genomes contain multiple RyR gene isoforms, expressed in different tissues and executing different functions. In contrast, invertebrate genomes contain a single RyR-encoding gene and it has long been proposed that different transcripts generated by alternative splicing may diversify their functions. Here, we analyze the expression and function of alternative exons in the C. elegans RyR gene unc-68. We show that specific isoform subsets are created via alternative promoters and via alternative splicing in unc-68 Divergent Region 2 (DR2), which actually corresponds to a region of high sequence variability across vertebrate isoforms. The expression of specific unc-68 alternative exons is enriched in different tissues, such as in body wall muscle, neurons and pharyngeal muscle. In order to infer the function of specific alternative promoters and alternative exons of unc-68, we selectively deleted them by CRISPR/Cas9 genome editing. We evaluated pharyngeal function, as well as locomotor function in swimming and crawling with high-content computer-assisted postural and behavioral analysis. Our data provide a comprehensive map of the pleiotropic impact of isoform-specific mutations and highlight that tissue-specific unc-68 isoforms fulfill distinct functions. As a whole, our work clarifies how the C. elegans single RyR gene unc-68 can fulfill multiple tasks through tissue-specific isoforms, and provide a solid foundation to further develop C. elegans as a model to study RyR channel functions and malfunctions.